Lac and lambda repressors relieve silencing of the Escherichia coli bgl promoter. Activation by alteration of a repressing nucleoprotein complex.

The Escherichia coli bgl promoter is kept in a repressed state by silencer sequences which flank the promoter and by the histone-like protein H-NS. Silencing of the bgl promoter is likely due to the formation of a repressing nucleoprotein complex of which H-NS is an essential component. Here, we show that silencing is abolished by the binding of Lac or lambda repressors to their respective operators that were inserted within the bgl upstream silencer. Efficient activation of bgl operon transcription by Lac and lambda repressors was independent of the position and phasing of the operators with respect to the promoter. Activation by Lac and lambda repressors as shown here is unprecedented. We conclude that the activation of bgl transcription by both repressors is achieved by a novel mechanism, that is by alteration of the repressing nucleoprotein complex rather than by protein-protein interactions with RNA polymerase and the catabolite activator protein, CAP.

[1]  O. Amster-Choder,et al.  Regulation of activity of a transcriptional anti-terminator in E. coli by phosphorylation in vivo. , 1990, Science.

[2]  B. Washburn,et al.  New method for generating deletions and gene replacements in Escherichia coli , 1989, Journal of bacteriology.

[3]  C. Gualerzi,et al.  Antagonistic involvement of FIS and H‐NS proteins in the transcriptional control of hns expression , 1996, Molecular microbiology.

[4]  A. Hochschild,et al.  Amino acid substitutions in the -35 recognition motif of sigma 70 that result in defects in phage lambda repressor-stimulated transcription , 1994, Journal of bacteriology.

[5]  M. De Felice,et al.  Cryptic operon for beta-glucoside metabolism in Escherichia coli K12: genetic evidence for a regulatory protein. , 1981, Genetics.

[6]  A. Bailone,et al.  Btcd, a mouse protein that binds to curved DNA, can substitute in Escherichia coli for H‐NS, a bacterial nucleoid protein. , 1996, The EMBO journal.

[7]  B. Müller-Hill.  The lac Operon: A Short History of a Genetic Paradigm , 1996 .

[8]  M. Mukerji,et al.  Transcriptional activation of the Escherichia coli bgl operon: negative regulation by DNA structural elements near the promoter , 1995, Molecular microbiology.

[9]  M Giel,et al.  A mutation in a new gene, bglJ, activates the bgl operon in Escherichia coli K-12. , 1996, Genetics.

[10]  C. Dorman,et al.  The StpA Protein Functions as a Molecular Adapter To Mediate Repression of the bgl Operon by Truncated H-NS in Escherichia coli , 1998, Journal of bacteriology.

[11]  L. Rasmussen,et al.  New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome , 1992 .

[12]  S. LeGrice,et al.  Enhancement of bacterial gene expression by insertion elements or by mutation in a CAP-cAMP binding site. , 1986, Journal of molecular biology.

[13]  H. Ingmer,et al.  H‐NS: a modulator of environmentally regulated gene expression , 1997, Molecular microbiology.

[14]  B. Kempf,et al.  Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli. , 1994, The Journal of biological chemistry.

[15]  J. Wang,et al.  Silencing of the Escherichia coli bgl promoter: effects of template supercoiling and cell extracts on promoter activity in vitro. , 1996, Nucleic acids research.

[16]  Andrew Wright,et al.  A bacterial gene involved in transcription antitermination: Regulation at a rho-independent terminator in the bgl operon of E. coli , 1987, Cell.

[17]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[18]  B. Hall,et al.  Mechanisms of activation of the cryptic cel operon of Escherichia coli K12. , 1990, Genetics.

[19]  Andrew Wright,et al.  Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific RNA binding protein , 1990, Cell.

[20]  R. Sternglanz,et al.  Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes , 1982, Cell.

[21]  Andrew Wright,et al.  Protein phosphorylation regulates transcription of the β-glucoside utilization operon in E. coli , 1989, Cell.

[22]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[23]  B. Uhlin,et al.  The positive regulator CfaD overcomes the repression mediated by histone‐like protein H‐NS (H1) in the CFA/I fimbrial operon of Escherichia coli. , 1992, The EMBO journal.

[24]  B. Rak,et al.  Regulation of the bgl operon of Escherichia coli by transcriptional antitermination. , 1988, The EMBO journal.

[25]  C. Higgins,et al.  In vivo supercoiling of plasmid and chromosomal DNA in an Escherichia coli hns mutant , 1997, Journal of bacteriology.

[26]  C. Gualerzi,et al.  The oligomeric structure of nucleoid protein H‐NS is necessary for recognition of intrinsically curved DNA and for DNA bending , 1997, The EMBO journal.

[27]  Ian R. Booth,et al.  A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli , 1988, Cell.

[28]  N. Goosen,et al.  Integration host factor alleviates the H‐NS‐mediated repression of the early promoter of bacteriophage Mu , 1996, Molecular microbiology.

[29]  H. Buc,et al.  Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H‐NS , 1994, Molecular microbiology.

[30]  T. Mizuno,et al.  Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS. , 1996, Journal of molecular biology.

[31]  C. Higgins,et al.  Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria , 1990, Cell.

[32]  A. Hochschild,et al.  Protein–Protein Contacts that Activate and Repress Prokaryotic Transcription , 1998, Cell.

[33]  A. Wright,et al.  Insertion of DNA activates the cryptic bgl operon in E. coli K12 , 1981, Nature.

[34]  B. Rak,et al.  High-level ribosomal frameshifting directs the synthesis of IS150 gene products. , 1991, Nucleic acids research.

[35]  M. Riley,et al.  The Bacterial Chromosome , 1990 .

[36]  M. Belfort,et al.  Nucleotide sequence of a newly-identified Escherichia coli gene, stpA, encoding an H-NS-like protein. , 1992, Nucleic acids research.

[37]  J. Wang,et al.  Transcription and DNA supercoiling. , 1993, Current opinion in genetics & development.

[38]  M. W. van der Woude,et al.  Thermoregulation of Escherichia coli pap transcription: H‐NS is a temperature‐dependent DNA methylation blocking factor , 1998, Molecular microbiology.

[39]  B. Rak,et al.  IS5: a mobile enhancer of transcription in Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  K. Schnetz,et al.  Silencing of Escherichia coli bgl promoter by flanking sequence elements. , 1995, The EMBO journal.

[41]  D. Ussery,et al.  DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression* , 1997, The Journal of Biological Chemistry.

[42]  O. Amster-Choder,et al.  Modulation of the dimerization of a transcriptional antiterminator protein by phosphorylation. , 1992, Science.

[43]  B. Rak,et al.  Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes , 1987, Journal of bacteriology.

[44]  M. Ptashne.  A genetic switch : phage λ and higher organisms , 1992 .

[45]  K. Timmis,et al.  Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors. , 1981, Gene.

[46]  R. Wagner,et al.  Evidence for a regulatory function of the histone‐like Escherichia coli protein H‐NS in ribosomal RNA synthesis , 1994, Molecular microbiology.

[47]  M. Susskind,et al.  Target of the transcriptional activation function of phage lambda cI protein. , 1994, Science.

[48]  Roberto Spurio,et al.  Expression of the gene encoding the major bacterial nucleoid protein H‐NS is subject to transcriptional auto‐repression , 1993, Molecular microbiology.

[49]  T. Mizuno,et al.  The leuO Gene Product Has a Latent Ability To Relieve bgl Silencing inEscherichia coli , 1998, Journal of bacteriology.

[50]  Richard H. Ebright,et al.  Promoter structure, promoter recognition, and transcription activation in prokaryotes , 1994, Cell.

[51]  S. Naitza,et al.  H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome , 1995, Journal of bacteriology.

[52]  D. Ussery,et al.  The chromatin-associated protein H-NS. , 1994, Biochimie.

[53]  C. Higgins,et al.  Expression and mutational analysis of the nucleoid‐associated protein H‐NS of Salmonella typhimurium , 1992, Molecular microbiology.

[54]  A. Spassky,et al.  Sequence determinants for H1 binding on Escherichia coli lac and gal promoters. , 1990, Biochemistry.

[55]  D. Ussery,et al.  The chromatin‐associated protein H‐NS alters DNA topology in vitro. , 1994, The EMBO journal.

[56]  M. Mukerji,et al.  Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H‐NS, and CRP–cAMP in regulation , 1997, Molecular microbiology.

[57]  C. Higgins,et al.  The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression , 1992, Cell.